DNA quality assessments using repetitive sequences

ABSTRACT

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 15/919,640, filed Mar. 13, 2018, now issued as U.S.Pat. No. 10,316,351 on Jun. 11, 2019, which is a continuation of U.S.application Ser. No. 14/208,460, filed Mar. 13, 2014, now issued as U.S.Pat. No. 9,963,736 on May 8, 2018, which claims priority to U.S.Provisional Application No. 61/783,225 filed Mar. 14, 2013. U.S.application Ser. No. 15/919,640 is herein incorporated by reference inits entity.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 830109_404C2_SEQUENCE_LISTING.txt. The text fileis 5 KB, was created on Apr. 19, 2019, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

The present disclosure relates to methods, arrays and kits for assessingthe quality of DNA samples.

Description of the Related Art

DNA has become biomarkers with the most potential. Compared to otherbiomarkers, DNA is stable and has less dynamic change. In addition, DNAas biomarkers is sequence based and has less ambiguity. Recently,discovery of DNA as biomarkers has been propelled by the growingapplication of next generation sequencing (NGS).

The quality of any scientific data is directly proportional to that ofthe starting samples. It is important to assess the quality of astarting DNA sample for downstream molecular analyses, such as qPCRassays and NGS. A bad quality sample will generate unusable data, whichcauses waste in material and labor. Not knowing the DNA quality can alsolead to misuse of precious sample, which once used, cannot be recovered.

Various methods are known for DNA quality evaluation. Most commonly usedspectrometrical methods can address some of the questions in DNAconcentration and contaminants. However, it cannot detect manycontaminants easily or evaluate the actual impact of contaminants in themolecular assays. Electrophoresis can evaluate the sizes of DNA sample.However, it cannot evaluate base damages, cross-linkage, modifications,which can adversely affect downstream enzymatic assays. Other peopleselect to test a group of gene assays in PCR to estimate actual sampleperformance in molecular assays. However, selection of a few gene assaysis sometimes biased, affected by biological changes in local chromosomalstructure (as seen in pathological conditions) and does not have broaderrepresentations. Thus, the prediction for a performance of DNA samplesin downstream molecular applications has been challenging, especiallyfor DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples.Accordingly, there is a need to establish an objective method toevaluate the quality of DNA for downstream DNA analyses.

SUMMARY

In one aspect, the present disclosure provides a method for assessingthe quality of a test genomic DNA sample, comprising:

(a) performing one or more real-time PCR reactions that use genomic DNAin a test genomic DNA sample as templates in the presence of one or moreprimer pairs, wherein each of the one or more primer pairs is specificfor amplifying identical or nearly identical genomic DNA fragments thatare present at multiple locations in the genome of the organism fromwhich the DNA sample is obtained,

(b) performing one or more real-time PCR reactions that use genomic DNAin a control genomic DNA sample as templates in the presence of the oneor more primer pairs used in step (a),

(c) determining the Ct values for the one or more real-time PCRreactions in step (a), and

(d) determining the Ct values for the one or more real-time PCRreactions in step (b),

wherein the difference between the Ct values determined in step (c) andthe corresponding Ct values determined in step (d) for the one or morereal-time PCR reactions are indicative of the quality of the testgenomic DNA sample.

In certain embodiments, the number of the primer pairs is 4-8.

In certain embodiments, the genomic DNA fragments amplified in thepresence of each primer pair are present at 10 or more differentlocations in the genome of the organism from which the DNA sample isobtained.

In certain embodiments, the genomic DNA fragments amplified in thepresence of each primer pair in combination are present in more than 80%of all autosomes of the organism from which the DNA sample is obtained.

In certain embodiments, the test genomic DNA sample is obtained fromhuman cells or tissue.

In certain embodiments, the test genomic DNA sample is obtained from aclinical sample.

In certain embodiments, the test genomic DNA sample is obtained from aformalin fixed and paraffin-embedded (FFPE) sample.

In certain embodiments, the genomic DNA fragments amplified in step (a)are between about 100 to 400 bp in length.

In certain embodiments, the genomic DNA fragments amplified in step (a)are of at least 2 substantially different sizes.

In certain embodiments, multiple real-time PCR reactions are performedin each of steps (a) and (b), and the average difference between the Ctvalues determined in step (c) and the corresponding Ct values determinedin step (d) for two or more of the multiple real-time PCR reactions isused to assess the quality of the test genomic DNA sample.

In certain embodiments, the primer pairs are selected from the followingprimer pairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ IDNOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ IDNOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9)SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, and (11) SEQ ID NOS:21and 22, (12) SEQ ID NOS:23 and 24.

In certain embodiments, the method further comprises performingadditional real-time PCR and/or NGS analysis of the test genomic DNAsample.

In another aspect, the present disclosure provides an array forassessing the quality of a test genomic DNA sample, comprising a solidsupport and multiple compartments in the solid support, wherein a firstprimer pair specific to a first genomic DNA fragment in the test genomicDNA sample is contained in a first compartment or each of a first set ofcompartments, and wherein (a) the first genomic DNA fragment and (b) oneor more fragments nearly identical to the first genomic DNA fragment, ifpresent in the genome of the organism from which the DNA sample isobtained are located at multiple sites in the genome.

In certain embodiments, the array further comprises a second compartmentor a second set of compartments, wherein a second primer pair specificto a second genomic DNA fragment in the test genomic DNA is contained inthe second compartment or each of the second set of compartments, andwherein (a) the second genomic DNA fragment and (b) one or morefragments nearly identical to the second genomic DNA fragment, ifpresent in the genome of the organism from which the DNA sample isobtained are located at multiple sites in the genome.

In certain embodiments, the array further comprises a third compartmentor a third set of compartments, wherein a third primer pair specific toa third genomic DNA fragment in the test genomic DNA is contained in thethird compartment or each of the third set of compartments, and wherein(a) the third genomic DNA fragment and (b) one or more fragments nearlyidentical to the third genomic DNA fragment, if present in the genome ofthe organism from which the DNA sample is obtained are located atmultiple sites in the genome.

In certain embodiments, the array further comprises a fourth compartmentor a fourth set of compartments, wherein a fourth primer pair specificto a fourth genomic DNA fragment in the test genomic DNA is present inthe fourth compartment or each of the fourth set of compartments, andwherein (a) the fourth genomic DNA fragment and (b) one or morefragments nearly identical to the fourth genomic DNA fragment, ifpresent in the genome of the organism from which the DNA sample isobtained are located at multiple sites in the genome.

In certain embodiments, the first, second, third, and fourth primerpairs if present in the array are selected from the following primerpairs: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ IDNOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9)SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, and (11) SEQ ID NOS:21and 22, (12) SEQ ID NOS:23 and 24.

In another aspect, the present disclosure provides a kit for assessingthe quality of a test genomic DNA sample, comprising: one or more primerpairs specific to one or more genomic DNA fragments in a test genomicDNA sample, wherein for each of the one or more genomic DNA fragments,(a) the genomic DNA fragment itself and (b) one or more fragments nearlyidentical to the genomic DNA fragment, if present in the genome of theorganism from which the test genomic DNA sample is obtained, are locatedat multiple sites in the genome.

In certain embodiments, the number of the primer pairs is 4 to 8.

In a related aspect, the present disclosure provides a kit for assessingthe quality of a test genomic DNA sample, comprising the array providedherein.

In certain embodiments, the kit further comprises a control genomic DNAsample.

In certain embodiments, the kit further comprises one or more reagentsfor performing real-time PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a DNA quality control PCR array plate layout.

FIG. 2 shows correlation between results from DNA Quality Control (QC)Panel analysis and NGS sequencing results. FFPE samples that generatedsuccessful sequencing data are indicated by empty bars and those thatdid not are shown by solid bars.

FIG. 3 shows the FFPE-1 sample's amplicon bias toward smaller read. Theleft graph is a read length histogram of control genomic DNA, and theright graph is a read length histogram of the FFPE-1 sample.

DETAILED DESCRIPTION

The present disclosure provides methods, arrays and kits for assessingthe quality of genomic DNA samples for downstream DNA analyses. Themethods provided herein analyze regions in the genome from which agenomic DNA sample is obtained that have identical or nearly identicalcopies randomly distributed across multiple or all autosomes. Bydesigning PCR assays for such regions, these methods assess sample DNAquality at many different chromosomal locations, and the performance ofsuch PCR assays is thus less affected by genomic heterogeneity in thegenomic DNA population in the sample. In addition, the PCR assays mayoptionally be designed to generate amplicons of different sizes. Bycomparing the performance of such PCR assays, the distribution ofamplifiable fragment in the genomic DNA sample may be evaluated. Thisinformation can help design the optimal molecular assay for a samplewith suboptimal quality.

In the following description, any ranges provided herein include all thevalues in the ranges unless otherwise indicated.

It should also be noted that the term “or” is generally employed in itssense including “and/or” (i.e., to mean either one, both, or anycombination thereof of the alternatives) unless the content clearlydictates otherwise.

Also, as used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise.

As used herein, “about” means±15% of the indicated range or value unlessotherwise indicated.

Genomic DNA Samples

The term “DNA” refers to a polymer comprising deoxyribonucleosides thatare covalently bonded, typically by phosphodiester linkages betweensubunits. This term includes but is not limited to genomic DNA (DNA inthe genome of an organism), cDNA (DNA reversely transcribed from mRNA),and plasmid DNA.

A “genomic DNA sample” is a sample that comprises genomic DNA isolatedfrom a source of interest.

Genomic DNA samples whose quality may be assessed by the methodsprovided herein include genomic DNA samples prepared from any samplesthat comprise genomic DNA. Exemplary samples from which genomic DNAsamples may be prepared include, but are not limited to, blood, swabs,body fluid, tissues including but not limited to, liver, spleen, kidney,lung, intestine, brain, heart, muscle, pancreas, cell cultures, planttissues or samples, as well as lysates, extracts, or materials andfractions obtained from the samples described above or any cells andmicroorganisms and viruses that may be present on or in a sample and thelike.

Materials obtained from clinical or forensic settings that containnucleic acids are also within the intended meaning of the term “sample”from which a genomic DNA sample may be prepared. Preferably, the sampleis a biological sample derived from a human, animal, plant, bacteria orfungi. The term “sample” also includes processed samples includingpreserved, fixed and/or stabilized samples, such as formalin fixed andparaffin-embedded (FFPE samples) and other samples that were treatedwith cross-linking fixatives such as glutaraldehyde.

Genomic DNA samples whose quality may be assessed by the methodsprovided herein may be prepared from nucleic acid-containing samples byany methods known in the art. Exemplary methods include lysis of nucleicacid-containing samples followed by isolating genomic DNA using, forexample, organic solvent such as phenol or nucleic acid binding columns.Genomic DNA samples may also be treated with RNase to reduce oreliminate RNA contamination.

Downstream Analyses

The methods for assessing the quality of genomic DNA samples providedherein are useful in determining whether a particular genomic DNA sampleis suitable for downstream molecular analyses, including NGS andreal-time PCR assays. Thus, various methods for assessing the quality ofgenomic DNA samples provided herein may further comprise performing oneor more additional analyses of the genomic DNA samples whose quality hasbeen assessed as suitable for such analysis. As described in detailbelow, assessing the quality of a genomic DNA sample includesdetermining amplification efficiency and size distribution ofamplifiable fragments of the sample.

Methods for Assessing Genomic DNA Quality

The present disclosure provides a method for assessing the quality of atest genomic DNA sample, comprising: (a) performing one or morereal-time PCR reactions that use genomic DNA in a test genomic DNAsample as templates in the presence of one or more primer pairs, whereineach of the one or more primer pairs is specific for amplifyingidentical or nearly identical genomic DNA fragments that are present atmultiple locations in the genome of the organism from which the DNAsample is obtained, (b) performing one or more real-time PCR reactionsthat use genomic DNA in a control genomic DNA sample as templates in thepresence of the one or more primer pairs used in step (a), (c)determining the Ct values for the one or more real-time PCR reactions instep (a), and (d) determining the Ct values for the one or morereal-time PCR reactions in step (b), wherein the difference between theCt values determined in step (c) and the corresponding Ct valuesdetermined in step (d) for the one or more real-time PCR reactions areindicative of the quality of the test genomic DNA sample.

The quality of a test genomic DNA sample refers to characteristics ofthe test genomic DNA sample related to downstream analyses of thesample, such as genomic DNA concentration, presence of contaminants,genomic DNA sizes, degree of degradation, presence of base damages,cross-linkage, or modifications, amplification efficiency, sizedistribution of amplifiable fragments, and the like.

A method for assessing the quality of a test genomic DNA sample providedherein may assess one or more characteristics of the test genomic DNAsample. For example, the method may be able to characterize theamplification efficiency of a test genomic DNA sample, and thus suggestappropriate amounts of the samples to be used in downstream analyses.The method may also be able to characterize the size distribution ofamplifiable fragments, which may guide the design of downstream analysesto achieve optimal use of the sample.

The method provided herein uses one or more primer pairs each of whichis specific for amplifying identical or nearly identical genomic DNAfragments that are present at multiple locations in the genome of theorganism from which the genomic DNA sample is obtained. The presence ofthe identical or nearly identical genomic DNA fragments at multiplelocations in the genome provides a broader representation of the genomicDNA population in the genomic DNA sample than a genomic DNA fragmentpresent only at one location in the genome. Thus, the overallamplification performance using such primer pair(s) is less affected bygenomic heterogeneity in the genomic DNA population of the sample.

For example, in the Example described below, the primer pair for PrimerAssay No. 1 (SEQ ID NOS:1 and 2) is able to amplify a 111 bp genomic DNAfragment that is present in 20 locations in the human genome and another110 bp nearly identical sequence at another location in the humangenome. Thus, using this primer pair, genomic DNA amplification at 21different locations in the human genome may be analyzed.

A genomic DNA fragment is nearly identical to another DNA if (a) thesize difference between the two genomic DNA fragments is at most 5%(e.g., at most 4%, 3%, 2% or 1%) of the full length of the longerfragment, and (b) the sequence identity between the two fragments is atleast 95% (e.g., at least 96%, 97%, 98%, or 99%).

Preferably, genomic DNA fragments amplified in the presence of a primerpair only have at most 4% differences in size and in sequence betweeneach other. More preferably, genomic DNA fragments amplified in thepresence of a primer pair only have at most 2% differences in size andin sequence between each other.

While any method known in the art for making such determinations may beused, for the purpose of the present invention, the BLAST algorithmdescribed in Altschul et al., J. Mol. Biol. 215:403-410 (1990) andKarlin et al., PNAS USA 90:5873-5787 (1993) is used for determiningsequence identity according to the methods of the invention. Aparticularly useful BLAST program is the WU-BLAST-2 program (Altschul etal., Methods in Enzymology 266:460-480 (1996)). WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A percent nucleic acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

An “oligonucleotide” refers to a short polymer composed ofdeoxyribonucleotides, ribonucleotides or combinations thereof.Oligonucleotides are generally between about 10 to 100 nucleotides,preferably about 15 to 30 nucleotides, in length.

A “primer” for amplification is an oligonucleotide that is complementaryto a target nucleotide sequence and leads to addition of nucleotides tothe 3′ end of the primer in the presence of a DNA or RNA polymerase.

A primer pair “specific for amplifying” a target sequence or a primerpair “specific to” a target sequence refers to a primer pair capable ofspecifically amplifying the target sequence.

“Specifically amplifying” a target sequence means amplifying the targetsequence or a sequence that is nearly identical to the target sequencewithout amplifying other sequences in a reaction mixture.

A sequence that is “nearly identical” to a target sequence if the sizedifference between these two sequences are at most 5% of the longerfragment and the sequence identity between these two sequences is atleast 95%.

The genomic DNA fragments amplified in the presence of a particularprimer pair may be present at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more different locations in agenome of interest. They may be present at 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore different autosomes. Preferably, no more than 50%, 40%, 30% or 20%of the genomic DNA fragment amplified by the particular primer pair arelocated on a single chromosome.

The genomic DNA fragments amplified by multiple primer pairs may bepresent in more than 50%, 60%, 70%, 80%, 85%, 90%, or 95% of allautosomes of the organism from which a genomic DNA sample is obtained.Preferably, the genomic DNA fragments amplified are not present on sexchromosomes. Also preferably, no more than 50%, 40%, 30% or 20% of thegenomic DNA fragment amplified by the multiple primer pairs are locatedon a single chromosome.

Primer pairs used in the method provided herein may be designed byidentifying regions in a genome of interest regions that have identicalor highly nearly identical copies distributed on different chromosomesusing bioinformatic approach. Preferably, such regions do not containknown single nucleotide polymorphisms (SNPs), insertions or deletions(INDELs), repetitive sequences, or other variations.

The method provided herein may use 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or more primer pairs.

In one embodiment, two primer pairs may share a primer so that theshorter amplicons produced by one primer pair are completely within thelonger amplicons produced by the other primer pair. This configurationallows evaluation of the frequency of random DNA damages occurred in thesample.

The genomic DNA fragments amplified by real-time PCR may be from about60 to 600 bp, such as about 80, 100, 120, 140, 160, 180, 200, 220, 240,260, 280, 300, 320, 340, 360, 380, 400, 450, 500, or 550 bp.

The sizes of the genomic DNA fragments amplified by different primerpairs may be the same or different. For example, a method providedherein may use primer pairs that generate amplicons that are about 100,200, 300 and 400 bp. It is also contemplated that multiple primer pairsare used to produce amplicons of the same or a similar size. Forexample, the method in the Example used 12 primer pairs: a first set of3 primer pairs amplified genomic DNA fragments of about 100 bp, a secondset of 3 primer pairs amplified genomic DNA fragments of about 200 bp, athird set of 3 primer pairs amplified genomic DNA fragments of about 300bp, and a fourth set of 3 primer pairs amplified genomic DNA fragmentsof about 400 bp. The use of primer pairs that amplify genomic DNAfragments of different sizes allows the evaluation of the distributionof amplifiable fragments in a genomic DNA sample, which in turn guidesthe best use of samples of inferior quality.

To ensure broad applicability of the method provided herein, the primerpairs may be tested to assess population variability. For example, ifthe method is for assessing the quality of human genomic DNA samples,candidate primer pairs may be used to analyze genomic DNA samples fromdifferent human populations to evaluate their consistency. If the methodis for assessing the quality of mouse genomic DNA samples, candidateprimer pairs may be used to analyze genomic DNA samples from differentmouse strains.

In one embodiment, the method provided herein is to assess the qualityof genomic DNA samples prepared from samples that contain human cells ortissues. The method may use one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12) of the following primer pairs: (1) SEQ ID NOS:1 and2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ IDNOS:19 and 20, (11) SEQ ID NOS:21 and 22, and (12) SEQ ID NOS:23 and24). The sequences of such primer pairs are provided in the Examplebelow.

When multiple primer pairs are used, each primer pair may be included inan individual real-time PCR reaction. Alternatively, multiple primerpairs may be included in a single real-time PCR reaction. For example,some primer pairs may be included in a single PCR reaction, and theother primer pairs may be included in one or more other PCR reactions.In one embodiment, two or more PCR primers that amplify genomic DNAfragments of the same or a similar size are included in a single PCRreaction. A genomic DNA fragment is of a similar size as another genomicDNA fragment if the difference between the two fragments is less than 5%(e.g., less than 4%, 3%, 2% or 1%) of the longer fragment.

Preferably, the test genomic DNA sample is from a sample that compriseshuman cells or tissue, such as a clinical sample. Exemplary preferredprimer pairs for assessing the quality of human genomic DNA samples areprovided in the Example.

A control genomic DNA sample is a genomic DNA sample isolated from asample that contains cells or tissue from the same species as the samplefrom which a test genomic DNA sample is isolated. In addition, thecontrol genomic DNA sample has been shown to be of high quality by, forexample, real-time PCR, NGS or other analyses.

For example, if a method is to assess the quality of certain genomic DNAsamples prepared from human FFPF tissues, a control genomic DNA samplemay be a human genomic DNA sample prepared from human blood and has beenshown to be of good quality (e.g., with high amplification efficiencyand producing high quality NGS data).

“Real-time polymerase chain reaction (PCR)” (also referred to as “qPCR”)refers to a type of PCR that amplifies and simultaneously quantify atarget DNA molecule. Its key feature is that the amplified DNA isdetected as the reaction progresses in real time.

Similar to traditional PCR reactions, real-time PCR reaction mixturescontain DNA polymerase, such as Taq DNA polymerase (e.g., hot-start TaqDNA polymerase), buffer, magnesium, dNTPs, and optionally other agents(e.g., stabilizing agents such as gelatin and bovine serum albumin). Inaddition, real-time PCR reaction mixtures also contain reagents for realtime detection and quantification of amplification products.

For real-time detection and quantification, probes specific toamplification products may be detectably labeled with a fluorophore.Alternatively, the amplification reaction may be performed in thepresence of an intercalating dye. Changes in fluorescence during theamplification reaction are monitored and are used in measuring theamount of amplification products.

Exemplary fluorophore-labeled probes include TAQMAN® probes. Such aprobe is typically labeled with a reporter molecule such as afluorescent dye at its 5′ end and a quencher molecule at its 3′ end. Theclose proximity of the reporter molecule to the quencher moleculeprevents detection of its fluorescence. Breakdown of the probe by the 5′to 3′ exonuclease activity of a DNA polymerase (e.g., Taq polymerase)breaks the reporter-quencher proximity and thus allows unquenchedemission of fluorescence, which can be detected. An increase in theproduct targeted by the reporter probe at each PCR cycle thereforecauses a proportional increase in fluorescence due to the breakdown ofthe probe and release of the reporter. Additional exemplaryfluorophore-labeled probes similar to TAQMAN® probes include MolecularBeacon probes and Scorpion probes.

Exemplary intercalating dyes include SYBR® Green. Such a dye binds toall double-stranded DNA in PCR, causing fluorescence of the dye. Anincrease in DNA product during PCR thus leads to an increase influorescence intensity and is measured at each cycle, thus allowing DNAconcentration to be quantified.

DNA quantification by real-time PCR may rely on plotting fluorescenceagainst the number of cycles on a logarithmic scale. A threshold fordetecting DNA-based fluorescence is set slightly above background. Thenumber of cycles at which the fluorescence exceeds the threshold iscalled “the threshold cycle (Ct).”

Preferably, one or more real-time PCR reactions that use genomic DNA ina test genomic DNA sample as templates performed in step (a) of themethod provided herein are performed under the same or similarconditions as the real-time PCR reactions that use genomic DNA in acontrol genomic DNA sample as templates are performed in step (b). Forexample, the real-time PCR reactions of steps (a) and (b) may beperformed in reaction mixtures containing the same DNA polymerase in thesame amount, the same PCR buffer, the same magnesium concentration, thesame intercalating dye in the same amount if the intercalating dye isused for detection and quantification of amplification products, and thesame dNTPs concentration and under the same thermocycling scheme.

Also preferably, the Ct values for the one or more real-time PCRreactions in step (a) determined in step (c) of the method providedherein are determined under the same or similar conditions as the Ctvalues for the one or more real-time PCR reactions in step (b) aredetermined in step (d). For example, the Ct determination in steps (c)and (d) may be performed using the same apparatus.

A Ct value determined in step (d) “corresponds to” a Ct value determinedin step (c) if the Ct value determined in step (d) is obtained from areaction mixture that contains the same primer pair or the same set ofprimer pairs as the reaction mixture from which the Ct value determinedin step (c) is obtained. In other words, the Ct value determined in step(c) and the “corresponding” Ct value determined in step (d) are toquantify genomic DNA fragments amplified in the presence of the sameprimer pair or the same set of primer pairs.

The difference between a Ct value determined in step (c) and thecorresponding Ct value determined in step (d) may be used tocharacterize or indicate the quality of the test genomic DNA sample. Forexample, the difference equal to or less than a predetermined value mayindicate that the test genomic DNA sample is of high quality that issuitable for downstream analysis (e.g., qPCR and NGS), whereas thedifference more than a predetermined value may indicate that the testgenomic DNA sample is of low quality. The predetermined value for agiven primer pair may be obtained using control samples and othersamples for which downstream analyses have been performed.

If a method for assessing the quality of a test genomic DNA samplecomprises amplifying genomic DNA fragments using multiple primer pairs,the Ct difference between real-time PCR reactions using genomic DNA in atest genomic DNA sample as templates and those using genomic DNA in acontrol genomic DNA sample as templates for each primer pair may bedetermined and used to characterize or indicate the quality of the testgenomic DNA sample. In addition, the average of the Ct differences forthe multiple primer pairs may be used to characterize or indicate theoverall quality of the test genomic DNA sample.

The method for assessing the quality of a test genomic DNA sample maycomprise amplifying genomic DNA fragments using multiple primer pairsthat produce amplicons of the same or a substantially similar size butwith substantially different sequences. Amplicons are similar in size ifthe size differences among these amplicons are less than about 25% ofthe longest amplicon. Amplicons are substantially different in sequencesif sequence identities among these amplicons are less than about 50%. Insuch a case, the Ct difference between real-time PCR reactions usinggenomic DNA in a test genomic DNA sample as templates and those usinggenomic DNA in a control genomic DNA sample as templates for each primerpair may be determined and used to characterize or indicate the qualityof the test genomic DNA sample. In addition, the average of the Ctdifferences for the multiple primer pairs may be used to characterize orindicate the quality of the test genomic DNA sample with respect toamplifying genomic DNA fragments that are similar in size as thoseamplified using the primer pairs. If the method also uses other primerpairs that amplify genomic DNA fragments that are substantiallydifferent in size from those described above, the average of the Ctdifferences for all the primer pairs used may be determined and used tocharacterize or indicate the overall quality of the test genomic DNAsample.

The method for assessing the quality of a genomic DNA sample may usemultiple primer pairs that amplify genomic DNA fragments having at least2, 3, 4, or 5 substantially different sizes. Two genomic DNA fragmentsare substantially different in size if their size different is more than50 bp, such as more than 75 bp, 100 bp, 125 bp, or 150 bp. For example,in the method described below in the Example, 12 primer pairs were usedto produce genomic DNA fragments having 4 substantially different sizes:about 100 bp, about 200 bp, about 300 bp and about 400 bp.

Arrays for Assessing Genomic DNA Quality

The present disclosure also provides an array for assessing the qualityof a DNA sample.

The array comprises a solid support and multiple compartments in thesolid support. Exemplary arrays include multi-well plates, such as96-well, 100-well, 384-well plates, and the like. The layout of anexemplary array is shown in FIG. 1.

The array provided herein may comprise a first primer pair specific to afirst genomic DNA fragment in a test genomic DNA sample in a firstcompartment or each of a first set of compartments, and wherein (a) thefirst genomic DNA fragment and (b) one or more fragments nearlyidentical to the first genomic DNA fragment, if present in the genome ofthe organism from which the DNA sample is obtained are located atmultiple sites in the genome.

The array may further comprise a second compartment or a second set ofcompartments, wherein a second primer pair specific to a second genomicDNA fragment in the test genomic DNA is contained in the secondcompartment or each of the second set of compartments, and wherein (a)the second genomic DNA fragment and (b) one or more fragments nearlyidentical to the second genomic DNA fragment, if present in the genomeof the organism from which the DNA sample is obtained are located atmultiple sites in the genome.

The array may further comprise a third compartment or a third set ofcompartments, wherein a third primer pair specific to a third genomicDNA fragment in the test genomic DNA is contained in the thirdcompartment or each of the third set of compartments, and wherein (a)the third genomic DNA fragment and (b) one or more fragments nearlyidentical to the third genomic DNA fragment, if present in the genome ofthe organism from which the DNA sample is obtained are located atmultiple sites in the genome.

The array may further comprise a fourth compartment or a fourth set ofcompartments, wherein a fourth primer pair specific to a fourth genomicDNA fragment in the test genomic DNA is present in the fourthcompartment or each of the fourth set of compartments, and wherein (a)the fourth genomic DNA fragment and (b) one or more fragments nearlyidentical to the fourth genomic DNA fragment, if present in the genomeof the organism from which the DNA sample is obtained are located atmultiple sites in the genome.

The array may further comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 15, 18, 20, and 24) additional compartments or one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, and 24)additional set of compartments that contain one or more (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, and 24) primer pairs. Each of theadditional primer pairs is able to amplify identical or nearly identicalgenomic DNA fragments that are present at multiple sites in the genomeof the organism from which the test genomic DNA sample is obtained.

If a set of compartments are present in an array that each contain thesame primer pair, one of the compartments may contain, in addition tothe primer pair, a portion of a control genomic DNA sample.

The primer pairs that may be included in the array provided herein andcontrol genomic DNA samples are described above with respect to methodsfor assessing the quality of a test genomic DNA sample.

In a related aspect, the present disclosure also provides use of thearray provided herein in assessing the quality of a genomic DNA sample,including determining suitability of using the genomic DNA sample indownstream analysis such as NGS and additional real-time PCR analysisand determining the size distribution of amplifiable fragments in thegenomic DNA sample.

Kits for Assessing Genomic DNA Quality

The present disclosure also provides kits for assessing the quality of agenomic DNA sample.

The kit provided herein comprises one or more primer pairs specific toone or more genomic DNA fragments in a test genomic DNA sample, whereinfor each of the one or more genomic DNA fragments, (a) the genomic DNAfragment itself and (b) one or more fragments nearly identical to thegenomic DNA fragment, if present in the genome of the organism fromwhich the test genomic DNA sample is obtained, are located at multiplesites in the genome. The number of the primer pairs in a kit may be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or more.

The one or more primer pairs may be in an array format. Thus, the kitprovided herein may comprise an array for assessing genomic DNA qualityas described above.

The kit may further comprise a control genomic DNA sample.

The kit may further comprise one or more reagents for performingreal-time PCR.

The reagents include buffer, magnesium, dNTPs, DNA polymerase (e.g., hotstart polymerase), and other agents (e.g., stabilizing agents such asgelatin and bovine serum albumin). Some of the reagents (e.g., dNTPs,magnesium, and buffer) may be pre-mixed to form a PCR reaction mix thatis included in the kit.

The primer pairs that may be included in the kit provided herein andcontrol genomic DNA samples are described above with respect to methodsfor assessing the quality of a test genomic DNA sample.

In a related aspect, the present disclosure also provides use of the kitprovided herein in assessing the quality of a genomic DNA sample,including determining suitability of using the genomic DNA sample indownstream analysis such as NGS and additional real-time PCR analysisand determining the size distribution of amplifiable fragments in thegenomic DNA sample.

The following example is for illustration and is not limiting.

EXAMPLE

Using bioinformatic approach, more than a few hundred regions in thehuman genome that have identical or highly similar copies randomlydistributed on different chromosomes were identified. Real-time PCRamplicons of approximately 100, 200, 300 and 400 bp were designed formost of them. Some shorter PCR amplicons were completely within otherlonger PCR amplicons, and two of the pairs share one common primer site.This configuration made it feasible to evaluate the frequency of randomDNA damages occurred in the sample.

The performance of those PCR assays was verified in over a hundrednormal human DNA samples representing different ethnic origins. 48assays were selected based on the assay sensitivity and robustness inthe tested population. Based on qPCR assay performance, 12 assays wereselected for further evaluation.

For each of the 12 assays, the primer pair sequences, the number ofsequences that the primer pair is able to amplify (referred to as“number of hits”) in the human genome, hg19 (GRCh37 Genome ReferenceConsortium Human Reference 37 (GCA_000001405.1)), the size of the firsthit, and the location of first hit are provided in Table 1 below:

TABLE 1 SEQ Assay Primer Sequence ID No. of First Hit No. (5′→3′) NO:Hits Size First Hit Location  1 TGGTAGCTTGAGTCACTGTG 1 21 111 bpchr11: 135345 + 135455 GGATTTGGGCATAGGTTTG 2  2 ATGATGGATCTTTCCCAAC 3 24103 bp chr15: 20140335 + 20140437 TGACAAGTAAAGCTGGAATAATC 4  3TAAATCATCCACATACTGAAGGAC 5 25  92 bp chr10: 66540447 + 66540538ATAGCCCTCATCTGTTTGGTC 6  4 TTCCCACACCAGTCTTCAC 7 22 205 bpchr11: 135251 + 135455 GGATTTGGGCATAGGTTTG 8  5 CCTCCCAAGTGTTCTGCTC 9 27212 bp chr15: 20140226 + 20140437 TGACAAGTAAAGCTGGAATAATC 10  6CCTTATTATCACCCTGCTCTC 11 31* 219 bp chr10: 99460360 + 99460578CCTGTGGGTATTTCTAGTCG 12  7 CCTCACTCCCTCACTCGAC 13 27 309 bpchr11: 135147 + 135455 GGATTTGGGCATAGGTTTG 14  8 TCACTCCCTCACTCGACAC 1527 307 bp chr11: 135149 + 135455 GGATTTGGGCATAGGTTTG 16  9TATAAAGGCACTAATCCCATTC 17 22 295 bp chr15: 20140175 + 20140469TTACATAGGACAGATGCAAATAGAC 18 10 TCATCTGAGAAGGTGGAGC 19 20 380 bpchr11: 135076 + 135455 GGATTTGGGCATAGGTTTG 20 11 CAAATTCAGTGTTGATGAGAGC21 26 399 bp chr15: 20140071 + 20140469 TTACATAGGACAGATGCAAATAGAC 22 12GCCTCGTGGGATGAGAAAG 23 57* 401 bp chr10: 99459976 + 99460376GCAGGGTGATAATAAGGAGAAG 24 *The numbers of hits for Assay Nos. 6 and 12do not include 2 hits in unplaced contigs for Assay No. 6 or 3 hits inunplaced contigs for Assay No. 12.

For each assay, the oligonucleotide in the top row in the above table isthe forward 5′-primer, whereas the oligonucleotide in the bottom row isthe reverse 3′-primer.

The locations and sizes of the hits (that have perfect matches with 19nucleotides at the 3′ end of primers) for each of the twelve primerpairs are provided in Tables 2-13 below.

TABLE 2 Targets for Primer Assay No. 1 Hit No. Hit Location (hg19) HitSize (bp) 1 chr11: 135345 + 135455 111 2 chr11: 131922 + 132032 111 3chr19: 205625 + 205735 111 4 chr19: 202211 + 202321 111 5 chr1:243221121 + 243221231 111 6 chr1: 675964 + 676074 111 7 chr1: 672548 +672658 111 8 chr1: 669142 + 669251 110 9 chr1: 665733 + 665843 111 10chr1: 140245 + 140355 111 11 chr7: 56901226 + 56901336 111 12 chr10:38736084-38736194 111 13 chr16: 90231916-90232026 111 14 chr16:90228497-90228607 111 15 chr1: 323780-323890 111 16 chr1: 320373-320483111 17 chr3: 197944687-197944797 111 18 chr3: 197941273-197941383 111 19chr5: 180750395-180750505 111 20 chr5: 180746988-180747098 111 21 chr7:128290584-128290694 111

TABLE 3 Targets for Primer Assay No. 2 Hit No. Hit Location (hg19) HitSize (bp) 1 chr15: 20140335 + 20140437 103 2 chr16: 34178086 + 34178188103 3 chr16: 33844143 + 33844245 103 4 chr16: 33061841 + 33061943 103 5chr16: 32118544 + 32118646 103 6 chr2: 92265099 + 92265201 103 7 chr2:91988683 + 91988785 103 8 chr2: 91661079 + 91661180 102 9 chr2:90439250 + 90439351 102 10 chr7: 64575049 + 64575151 103 11 chr7:61675244 + 61675346 103 12 chr9: 42734973 + 42735075 103 13 chr10:42604408-42604510 103 14 chr16: 32831850-32831952 103 15 chr21:10893340-10893442 103 16 chr2: 132804665-132804767 103 17 chr2:132765379-132765481 103 18 chr2: 92240041-92240143 103 19 chr7:65046135-65046237 103 20 chr7: 64983245-64983347 103 21 chr7:57937370-57937472 103 22 chr9: 70411890-70411992 103 23 chr9:70160962-70161064 103 24 chr9: 69795962-69796064 103

TABLE 4 Targets for Primer Assay No. 3 Hit No. Hit Location (hg19) HitSize (bp) 1 chr10: 66540447 + 66540538 92 2 chr11: 101107279 + 10110737092 3 chr12: 95847668 + 95847759 92 4 chr1: 94840246 + 94840337 92 5chr2: 218982552 + 218982643 92 6 chr3: 44588305 + 44588396 92 7 chr4:35862674 + 35862765 92 8 chr6: 87386987 + 87387078 92 9 chr7:151729234 + 151729325 92 10 chr7: 23897603 + 23897694 92 11 chr8:87942626 + 87942717 92 12 chr8: 54052020 + 54052111 92 13 chr8:42419179 + 42419270 92 14 chr10: 121647046-121647137 92 15 chr12:131823434-131823525 92 16 chr19: 8448392-8448483 92 17 chr1:161224222-161224313 92 18 chr1: 105655701-105655792 92 19 chr2:98441604-98441695 92 20 chr3: 190907594-190907685 92 21 chr3:164533317-164533408 92 22 chr4: 45537496-45537587 92 23 chr5:114330130-114330221 92 24 chr6: 38588227-38588318 92 25 chr6:16459961-16460052 92

TABLE 5 Targets for Primer Assay No. 4 Hit No. Hit Location (hg19) HitSize (bp) 1 chr11: 135251 + 135455 205 2 chr11: 131828 + 132032 205 3chr19: 205531 + 205735 205 4 chr19: 202117 + 202321 205 5 chr1:243221027 + 243221231 205 6 chr1: 675870 + 676074 205 7 chr1: 672454 +672658 205 8 chr1: 669048 + 669251 204 9 chr1: 665639 + 665843 205 10chr1: 140151 + 140355 205 11 chr7: 56446682 + 56446886 205 12 chr7:55816209 + 55816413 205 13 chr16: 90231916-90232120 205 14 chr16:90228497-90228701 205 15 chr1: 323780-323984 205 16 chr1: 320373-320577205 17 chr3: 197944687-197944891 205 18 chr3: 197941273-197941477 205 19chr5: 180750395-180750599 205 20 chr5: 180746988-180747192 205 21 chr7:128290584-128290788 205 22 chr7: 45846431-45846635 205

TABLE 6 Targets for Primer Assay No. 5 Hit No. Hit Location (hg19) HitSize (bp) 1 chr15: 20140226 + 20140437 212 2 chr16: 34177977 + 34178188212 3 chr16: 33844034 + 33844245 212 4 chr16: 33061732 + 33061943 212 5chr16: 32118435 + 32118646 212 6 chr2: 92264990 + 92265201 212 7 chr2:91988574 + 91988785 212 8 chr2: 91660970 + 91661180 211 9 chr2:90439141 + 90439351 211 10 chr7: 64574941 + 64575151 211 11 chr9:42734864 + 42735075 212 12 chr10: 42615575-42615786 212 13 chr10:42604408-42604618 211 14 chr16: 46469617-46469827 211 15 chr16:46458916-46459127 212 16 chr16: 32831850-32832061 212 17 chr21:10893340-10893551 212 18 chr2: 132804665-132804876 212 19 chr2:132765379-132765590 212 20 chr2: 92240041-92240252 212 21 chr7:65046135-65046345 211 22 chr7: 64983245-64983455 211 23 chr7:61761699-61761910 212 24 chr7: 57937370-57937581 212 25 chr9:70411890-70412101 212 26 chr9: 70160962-70161173 212 27 chr9:69795962-69796173 212

TABLE 7 Targets for Primer Assay No. 6 Hit No. Hit Location (hg19) HitSize (bp) 1 chr10: 99460360 + 99460578 219 2 chr10: 29716426 + 29716644219 3 chr12: 49510213 + 49510431 219 4 chr13: 99417362 + 99417580 219 5chrUn_gl000223: 15304 + 15518 215 6 chrUn_gl000223: 10365 + 10582 218 7chr1: 47599632 + 47599850 219 8 chr2: 95857874 + 95858092 219 9 chr4:9668931 + 9669149 219 10 chr4: 9132851 + 9133068 218 11 chr4: 9124292 +9124510 219 12 chr5: 56843254 + 56843466 213 13 chr6: 52748818 +52749037 220 14 chr8: 8063979 + 8064196 218 15 chr9: 28880975 + 28881187213 16 chrX: 153750154 + 153750366 213 17 chrX: 126488690 + 126488902213 18 chr11: 67597481-67597698 218 19 chr11: 3468681-3468898 218 20chr12: 133672083-133672296 214 21 chr12: 133667147-133667361 215 22chr13: 114947411-114947629 219 23 chr15: 90889818-90890036 219 24 chr2:169418786-169418998 213 25 chr3: 195454995-195455213 219 26 chr4:157307531-157307749 219 27 chr4: 3987662-3987874 213 28 chr4:3979076-3979294 219 29 chr5: 39828181-39828395 215 30 chr7:67568765-67568983 219 31 chr7: 38270470-38270688 219 32 chr8:12316517-12316734 218 33 chr8: 12073995-12074212 218

TABLE 8 Targets for Primer Assay No. 7 Hit No. Hit Location (hg19) HitSize (bp) 1 chr11: 135147 + 135455 309 2 chr11: 131724 + 132032 309 3chr19: 205427 + 205735 309 4 chr19: 202013 + 202321 309 5 chr1:243220923 + 243221231 309 6 chr1: 222650846 + 222651154 309 7 chr1:675766 + 676074 309 8 chr1: 672350 + 672658 309 9 chr1: 668944 + 669251308 10 chr1: 665535 + 665843 309 11 chr1: 140047 + 140355 309 12 chr4:120331644 + 120331952 309 13 chr7: 56446578 + 56446886 309 14 chr7:55816105 + 55816413 309 15 chr10: 38736084-38736392 309 16 chr16:90231916-90232224 309 17 chr16: 90228497-90228805 309 18 chr1:323780-324088 309 19 chr1: 320373-320681 309 20 chr3:197944687-197944995 309 21 chr3: 197941273-197941581 309 22 chr4:119550936-119551244 309 23 chr5: 180750395-180750703 309 24 chr5:180746988-180747296 309 25 chr7: 128290584-128290892 309 26 chr7:45846431-45846739 309 27 chr7: 39830686-39830993 308

TABLE 9 Targets for Primer Assay No. 8 Hit No. Hit Location (hg19) HitSize (bp) 1 chr11: 135149 + 135455 307 2 chr11: 131726 + 132032 307 3chr19: 205429 + 205735 307 4 chr19: 202015 + 202321 307 5 chr1:243220925 + 243221231 307 6 chr1: 222650848 + 222651154 307 7 chr1:675768 + 676074 307 8 chr1: 672352 + 672658 307 9 chr1: 668946 + 669251306 10 chr1: 665537 + 665843 307 11 chr1: 140049 + 140355 307 12 chr4:120331646 + 120331952 307 13 chr7: 56446580 + 56446886 307 14 chr7:55816107 + 55816413 307 15 chr10: 38736084-38736390 307 16 chr16:90231916-90232222 307 17 chr16: 90228497-90228803 307 18 chr1:323780-324086 307 19 chr1: 320373-320679 307 20 chr3:197944687-197944993 307 21 chr3: 197941273-197941579 307 22 chr4:119550936-119551242 307 23 chr5: 180750395-180750701 307 24 chr5:180746988-180747294 307 25 chr7: 128290584-128290890 307 26 chr7:45846431-45846737 307 27 chr7: 39830686-39830991 306

TABLE 10 Targets for Primer Assay No. 9 Hit No. Hit Location (hg19) HitSize (bp) 1 chr15: 20140175 + 20140469 295 2 chr16: 33061681 + 33061975295 3 chr16: 32118384 + 32118678 295 4 chr2: 91660919 + 91661212 294 5chr2: 90439090 + 90439383 294 6 chr7: 64574890 + 64575183 294 7 chr7:61675084 + 61675378 295 8 chr9: 42734813 + 42735107 295 9 chr16:46469585-46469878 294 10 chr18: 15206512-15206806 295 11 chr18:15162997-15163291 295 12 chr21: 10893308-10893602 295 13 chr2:132804633-132804927 295 14 chr2: 132765347-132765641 295 15 chr7:65046103-65046396 294 16 chr7: 64983213-64983506 294 17 chr7:61761667-61761961 295 18 chr7: 61058228-61058522 295 19 chr7:57937338-57937632 295 20 chr9: 70411858-70412152 295 21 chr9:70160930-70161224 295 22 chr9: 69795930-69796224 295

TABLE 11 Targets for Primer Assay No. 10 Hit No. Hit Location (hg19) HitSize (bp) 1 chr11: 135076 + 135455 380 2 chr11: 131653 + 132032 380 3chr19: 205356 + 205735 380 4 chr19: 201942 + 202321 380 5 chr1: 675695 +676074 380 6 chr1: 672279 + 672658 380 7 chr1: 668873 + 669251 379 8chr1: 665464 + 665843 380 9 chr1: 139976 + 140355 380 10 chr7:56446507 + 56446886 380 11 chr7: 55816034 + 55816413 380 12 chr16:90231916-90232295 380 13 chr16: 90228497-90228876 380 14 chr1:323780-324159 380 15 chr1: 320373-320752 380 16 chr3:197944687-197945066 380 17 chr3: 197941273-197941652 380 18 chr5:180750395-180750774 380 19 chr5: 180746988-180747367 380 20 chr7:45846431-45846810 380

TABLE 12 Targets for Primer Assay No. 11 Hit No. Hit Location (hg19) HitSize (bp) 1 chr15: 20140071 + 20140469 399 2 chr16: 33061577 + 33061975399 3 chr16: 32118280 + 32118678 399 4 chr2: 92264835 + 92265233 399 5chr2: 91988419 + 91988817 399 6 chr2: 91660815 + 91661212 398 7 chr2:90438986 + 90439383 398 8 chr7: 64574786 + 64575183 398 9 chr7:61674980 + 61675378 399 10 chr9: 42734709 + 42735107 399 11 chr10:42604376-42604773 398 12 chr16: 46469585-46469982 398 13 chr18:15206512-15206910 399 14 chr18: 15162997-15163395 399 15 chr21:10893308-10893706 399 16 chr2: 132804633-132805031 399 17 chr2:132765347-132765745 399 18 chr2: 92240009-92240407 399 19 chr7:65046103-65046500 398 20 chr7: 64983213-64983610 398 21 chr7:61761667-61762065 399 22 chr7: 61058228-61058626 399 23 chr7:57937338-57937736 399 24 chr9: 70411858-70412256 399 25 chr9:70160930-70161328 399 26 chr9: 69795930-69796328 399

TABLE 13 Targets for Primer Assay No. 12 Hit No. Hit Location (hg19) HitSize (bp) 1 chr10: 99459976 + 99460376 401 2 chr10: 98163398 + 98163798401 3 chr10: 33183786 + 33184184 399 4 chr11: 123150791 + 123151191 4015 chr11: 67689132 + 67689532 401 6 chr11: 37952518 + 37952918 401 7chr12: 49509829 + 49510229 401 8 chr13: 43285312 + 43285712 401 9 chr21:39945179 + 39945579 401 10 chr22: 17540425 + 17540825 401 11chrUn_gl000223: 14920 + 15320 401 12 chrUn_gl000223: 9981 + 10381 401 13chrUn_gl000231: 22879 + 23279 401 14 chr1: 236668200 + 236668600 401 15chr1: 47599249 + 47599648 400 16 chr1: 40937804 + 40938204 401 17 chr2:95857490 + 95857890 401 18 chr3: 75587273 + 75587673 401 19 chr4:122318082 + 122318482 401 20 chr4: 9753064 + 9753464 401 21 chr4:4076419 + 4076819 401 22 chr5: 161797510 + 161797907 398 23 chr6:52748434 + 52748834 401 24 chr7: 104789485 + 104789885 401 25 chr8:12423891 + 12424287 397 26 chr8: 8063597 + 8063995 399 27 chr9:131612897 + 131613292 396 28 chr9: 129479179 + 129479579 401 29 chr9:28880591 + 28880991 401 30 chrX: 153749772 + 153750170 399 31 chrX:90425667 + 90426067 401 32 chr10: 96957178-96957577 400 33 chr11:118879040-118879439 400 34 chr11: 71384933-71385333 401 35 chr11:67597682-67598080 399 36 chr11: 23114276-23114676 401 37 chr11:3468882-3469280 399 38 chr12: 133672280-133672680 401 39 chr12:133667345-133667745 401 40 chr12: 8584230-8584630 401 41 chr13:114947613-114948011 399 42 chr14: 106487687-106488087 401 43 chr15:98172559-98172959 401 44 chr15: 90890020-90890420 401 45 chr1:113359623-113360023 401 46 chr3: 142471114-142471514 401 47 chr3:98081627-98082027 401 48 chr3: 15187255-15187655 401 49 chr4:157307733-157308132 400 50 chr4: 43003876-43004276 401 51 chr5:154023434-154023835 402 52 chr5: 152177074-152177473 400 53 chr6:52812723-52813123 401 54 chr6: 43207456-43207860 405 55 chr7:67568967-67569365 399 56 chr8: 12316718-12317116 399 57 chr8:12074196-12074594 399 58 chr8: 7556947-7557347 401 59 chr8:7098403-7098803 401 60 chr8: 6984728-6985128 401

The locations and sizes of additional hits with less stringent criteria(i.e., having perfect matches with 15 nucleotides at the 3′ end ofprimers) for some of the twelve primer pairs are provided in Table 14below.

TABLE 14 Additional Targets for Primer Assays Additional Hit Size AssayNo. Hit No. Hit Location (hg19) (bp) 2 1 chr1: 149030745 + 149030847 1032 chr7: 53197939-53198041 103 3 1 chr16: 57432726 + 57432817 92 2 chr8:39738896 + 39738987 92 3 chrX: 111355904 + 111355995 92 4 chr17:50654589-50654680 92 5 chr17: 26042855-26042946 92 6 chr2:148072985-148073076 92 7 chr4: 185659639-185659730 92 8 chr5:99320377-99320468 92 5 1 chr21: 9475041 + 9475252 212 6 1 chr10:33184168 + 33184386 219 2 chr11: 123151175 + 123151393 219 3 chr12:8417305-8417523 219 4 chr15: 40936175-40936393 219 5 chr1:147181566-147181784 219 8 1 chr7: 56901030 + 56901336 307 2 chr7:51460780 + 51461085 306 9 1 chr7: 53197907-53198201 295 10 1 chr1:243220852 + 243221231 380 2 chr1: 222650775 + 222651154 380 3 chr4:120331573 + 120331952 380 4 chr7: 51460707 + 51461085 379 5 chr10:38736084-38736463 380 6 chr4: 119550936-119551315 380 7 chr7:128290584-128290963 380 12 1 chr2: 94434 + 94835 402 2 chr5: 24466249 +24466649 401 3 chrY: 10013901 + 10014301 401 4 chr10:102793517-102793917 401 5 chr11: 3416009-3416409 401 6 chr14:20709942-20710342 401 7 chr16: 10923540-10923940 401 8 chr2:702776-703176 401 9 chr3: 121330178-121330561 384 10 chr4:128360853-128361253 401 11 chr4: 122367400-122367800 401 12 chr4:3907275-3907674 400

Methods for Performing PCR Assay

Genomic DNA was extracted from human FFPE samples (normal human genomicDNA from Promega (Cat. No. G304X, 90% of the DNA is longer than 50 kb insize as measured by pulsed-field gel electrophoresis) was used ascontrol). 1-2 ng of genomic DNA was used in each PCR reaction. DNAQuality Control (QC) PCR Array and Qiagen RT² Real-Timer SYBR Green/ROXPCR Mix were used to perform PCR. The 12 primer pair assays of the DNAQC PCR Array were predispensed in 384-well PCR plate as layout in FIG.1.

Methods for Performing Next Generation Sequencing

Briefly, a multiplexed PCR assay that targeted 400 amplicons wasdeveloped for target enrichment. Primers were designed to avoid knownSNPs and repetitive sequences. 20 ng of normal human genomic DNA orgenomic DNA isolated from FFPE samples were evaluated for multiplexedPCR based target enrichment. PCR enriched samples were subjected to NGSlibrary construction with Ion Xpress Plus Fragment Library Kit. Afterquantification with GeneRead Library Quantification Array, template(library) dilution factor was determined for each sample. Appropriatelydiluted prepared NGS libraries were sequenced on Ion Torrent PGMsequencer according to Life Technologies' user guide.

In a preliminary study, DNA from seven FFPE samples of varying qualityand control Universal DNA were analyzed in duplicate by real-time PCRusing 12 selected qPCR primer assays. The average Ct values of all threesets of 100 bp, 200 bp, 300 bp and 400 bp amplicons were determined foreach DNA sample. The average Ct value of specific amplicon size for thecontrol DNA was subtracted from average Ct value of that amplicon sizefor FFPE DNA.

The resulting ΔCt values were plotted against the outcome of sequencingresults (FIG. 2). Samples with ΔCt values lower than 5, 10, 15 and 17with 100 bp, 200 bp, 300 bp and 400 bp amplicons, respectively, showedgood sequencing results (FIG. 2). This suggests that selected 12 qPCRprimer assays can be used to pre-qualify DNA samples for successfulsequencing results.

Five out of seven FFPE samples generated successful sequencing results.One sample (FFPE-1) generated smaller read length as compared to controluniversal DNA as well as other analyzed samples (FIG. 3), suggesting lowquality of starting DNA with a potential risk of amplicon bias. TheFFPE-7 sample showed insufficient amount of amplifiable librarymolecules upon qPCR based library quantification, after NGS librarypreparation, and did not process for sequencing.

Assuming that the higher the library dilution factor, the better thequality of DNA, samples with successful sequencing results were rankedfor DNA quality as shown in Table 15 below. In addition, the average Ctvalue for the 12 assays of DNA QC Array was used to rank the DNAquality, assuming that the lower the averaged Ct value, the higher thequality rank. Interestingly, quality ranks based on the library dilutionfactors and based on the DNA QC Array analysis are the same. Thissuggests that the DNA QC Array analysis may be useful for recommendingappropriate DNA input amount for NGS.

TABLE 15 Library dilution Quality rank based on Quality rank based onSamples factor dilution factor DNA QC Panel results FFPE-2 16.0 1 1FFPE-3 4.3 5 5 FFPE-4 10.5 4 4 FFPE-5 12.3 3 3 FFPE-6 14.8 2 2

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A method for assessing the quality of atest genomic DNA sample for a downstream molecular DNA analysis,comprising: (a) performing one or more real-time PCR reactions that usegenomic DNA in a test genomic DNA sample as templates in the presence oftwo or more primer pairs, wherein each of the two or more primer pairsis specific for amplifying identical or nearly identical genomic DNAfragments that are present at 10 or more different locations in thegenome of the organism from which the test genomic DNA sample isobtained, wherein a genomic DNA fragment is nearly identical to anotherDNA if (i) the size difference between the two genomic DNA fragments isat most 5% of the full length of the longer fragment, and (ii) thesequence identity between the two fragments is at least 95%, (b)determining the Ct values for the one or more real-time PCR reactions instep (a), (c) determining the difference (ΔCt) between the Ct valuesdetermined in step (b) and the corresponding control Ct values of one ormore real-time PCR reactions using genomic DNA in a control genomic DNAsample as templates in the presence of the two or more primer pairs usedin step (a), wherein the difference is indicative of the quality of thetest genomic DNA sample, and (d) performing one or more additionalmolecular analyses of the genomic DNA test sample whose quality has beenassessed as suitable for such analysis.
 2. The method of claim 1,wherein the method comprises: (i) measuring PCR amplifiable molecules inthe test genomic DNA sample using the difference in the Ct values (ΔCt);and/or determining the input amount of test genomic DNA for real-timePCR reactions based on the ΔCt.
 3. The method of claim 1, having one ormore of the following characteristics: i) the genomic DNA fragmentsamplified by multiple primer pairs are present in more than 50%, 60%,70% or more than 80% of all autosomes of the organism from which the DNAsample is obtained; ii) the genomic DNA fragments amplified are notpresent on sex chromosomes; iii) no more than 50 percent, 40 percent, 30percent or 20 percent of the genomic DNA fragment amplified by themultiple primer pairs are located on a single chromosome; iv) thegenomic DNA fragments amplified in step (a) are between about 100 to 400bp in length; v) the genomic DNA fragments amplified in the presence ofa particular primer pair are present at 3 or more different autosomes;and/or vi) the number of the primer pairs is 2-12 or 4-8.
 4. The methodof claim 1, wherein i) the test genomic DNA sample is obtained fromhuman cells or tissue, ii) the test genomic DNA sample is obtained froma clinical human cells or tissue sample, and/or iii) the test genomicDNA sample is obtained from a formalin fixed and paraffin-embedded(FFPE) sample.
 5. The method of claim 1, wherein the genomic DNAfragments amplified in step (a) are of at least 2 substantiallydifferent sizes, wherein two genomic DNA fragments are substantiallydifferent in size if their size difference is more than 50 bp or morethan 75 bp.
 6. The method according to claim 5, wherein the methodincludes determining amplification efficiency and amount or sizedistribution of amplifiable fragments of the sample.
 7. The methodaccording to claim 5, wherein the method comprises comparing theperformance of PCR assays generating amplicons of different sizes andevaluating the distribution of amplifiable fragments in the genomic DNAsample.
 8. The method of claim 1, wherein multiple real-time PCRreactions are performed in step (a), and the average difference betweenthe Ct values determined in step (b) and the corresponding control Ctvalues for two or more of the multiple real-time PCR reactions is usedto assess the quality of the test genomic DNA sample.
 9. The method ofclaim 1, wherein the two or more primer pairs are selected from theprimer pairs consisting of: (1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and4, (3) SEQ ID NOS:5 and 6, (4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and10, (6) SEQ ID NOS:11 and 12, (7) SEQ ID NOS:13 and 14, (8) SEQ IDNOS:15 and 16, (9) SEQ ID NOS:17 and 18, (10) SEQ ID NOS:19 and 20, (11)SEQ ID NOS:21 and 22, and (12) SEQ ID NOS:23 and
 24. 10. The method ofclaim 1, wherein the organism for which the test genomic DNA sample isobtained is human, and wherein the identical or nearly identical genomicDNA fragments of step (a) are selected from the targets with chromosomallocations shown in Tables 2-14.
 11. The method of claim 1, furthercomprising performing additional real-time PCR and/or NGS analysis ofthe test genomic DNA sample and wherein optionally, the input amount ofthe test genomic DNA sample in the additional real-time PCR isdetermined based on the difference between the Ct values determined instep (b) and the corresponding control Ct values for the one or morereal-time PCR reactions.
 12. The method according to claim 1, whereineach primer pair is included in an individual real-time PCR reaction orwherein multiple primer pairs are included in a single real-time PCRreaction.
 13. The method according to claim 1, wherein assessing thequality of a genomic DNA sample includes determining amplificationefficiency and amount or size distribution of amplifiable DNA fragmentsin the sample.
 14. The method of claim 1, wherein an array is used instep (a), wherein the array comprises a solid support and multiplecompartments in the solid support, wherein a first primer pair specificto a first genomic DNA fragment in the test genomic DNA sample iscontained in a first compartment or each of a first set of compartments,and wherein (a) the first genomic DNA fragment and (b) one or morefragments nearly identical to the first genomic DNA fragment, if presentin the genome of the organism from which the DNA sample is obtained, arelocated at 10 or more different locations in the genome of the organismfrom which the test genomic DNA sample is obtained, the array optionallyfurther comprising a second compartment or a second set of compartments,wherein a second primer pair specific to a second genomic DNA fragmentin the test genomic DNA is contained in the second compartment or eachof the second set of compartments, and wherein (a) the second genomicDNA fragment and (b) one or more fragments nearly identical to thesecond genomic DNA fragment, if present in the genome of the organismfrom which the DNA sample is obtained are located at 10 or moredifferent locations in the genome of the organism from which the testgenomic DNA sample is obtained, and wherein a genomic DNA fragment isnearly identical to another DNA if (i) the size difference between thetwo genomic DNA fragments is at most 5% of the full length of the longerfragment, and (ii) the sequence identity between the two fragments is atleast 95%.
 15. The method of claim 14, wherein the array furthercomprises a third compartment or a third set of compartments, wherein athird primer pair specific to a third genomic DNA fragment in the testgenomic DNA is contained in the third compartment or each of the thirdset of compartments, and wherein (a) the third genomic DNA fragment and(b) one or more fragments nearly identical to the third genomic DNAfragment, if present in the genome of the organism from which the DNAsample is obtained are located at multiple sites in the genome, thearray optionally further comprising also a fourth compartment or afourth set of compartments, wherein a fourth primer pair specific to afourth genomic DNA fragment in the test genomic DNA is present in thefourth compartment or each of the fourth set of compartments, andwherein (a) the fourth genomic DNA fragment and (b) one or morefragments nearly identical to the fourth genomic DNA fragment, ifpresent in the genome of the organism from which the DNA sample isobtained, are located at multiple sites in the genome.
 16. The method ofclaim 14, wherein the first primer pair and the second primer pair, ifpresent, in the array are selected from the primer pairs consisting of:(1) SEQ ID NOS:1 and 2, (2) SEQ ID NOS:3 and 4, (3) SEQ ID NOS:5 and 6,(4) SEQ ID NOS:7 and 8, (5) SEQ ID NOS:9 and 10, (6) SEQ ID NOS:11 and12, (7) SEQ ID NOS:13 and 14, (8) SEQ ID NOS:15 and 16, (9) SEQ IDNOS:17 and 18, (10) SEQ ID NOS:19 and 20, (11) SEQ ID NOS:21 and 22, and(12) SEQ ID NOS:23 and
 24. 17. The method of claim 1, wherein the two ormore primer pairs are comprised in a kit, and wherein optionally: (i)the number of the primer pairs is 2 to 12 or 4 to 8; (ii) the kitcomprises an array, wherein the array comprises a solid support andmultiple compartments in the solid support, wherein a first primer pairspecific to a first genomic DNA fragment in the test genomic DNA sampleis contained in a first compartment or each of a first set ofcompartments, and wherein (a) the first genomic DNA fragment and (b) oneor more fragments nearly identical to the first genomic DNA fragment, ifpresent in the genome of the organism from which the DNA sample isobtained, are located at 10 or more different locations in the genome ofthe organism from which the test genomic DNA sample is obtained, thearray optionally further comprising a second compartment or a second setof compartments, wherein a second primer pair specific to a secondgenomic DNA fragment in the test genomic DNA is contained in the secondcompartment or each of the second set of compartments, and wherein (a)the second genomic DNA fragment and (b) one or more fragments nearlyidentical to the second genomic DNA fragment, if present in the genomeof the organism from which the DNA sample is obtained are located at 10or more different locations in the genome of the organism from which thetest genomic DNA sample is obtained, and wherein a genomic DNA fragmentis nearly identical to another DNA if (i) the size difference betweenthe two genomic DNA fragments is at most 5% of the full length of thelonger fragment, and (ii) the sequence identity between the twofragments is at least 95%; (iii) the kit further comprises a controlgenomic DNA sample; and/or (iv) the kit further comprises one or morereagents for performing real-time PCR.
 18. The method of claim 3, havingcharacteristic v), wherein no more than 50 percent, 40 percent, 30percent or 20 percent of the genomic DNA fragment amplified by theparticular primer pair are located on a single chromosome.